The biological functions and activity of our genomes is not determined by linear DNA sequence information alone. To fit within the nucleus, DNA assembles with nucleosomes to form chromatin that coils into spatially defined territories that determine if genes are active or silent through poorly understood mechanisms. The local and global organization of chromatin are integrated in the nucleus to determine gene activity and genome function. To visualize the different structural scales of genome function in intact cells, ChromEM has been developed that exploits a cell permeable fluorescent small molecule that binds specifically to DNA and upon excitation paints the surface of chromatin with an osmiophillic polymer that can be visualized using electron microscopy (EM). By combining ChromEM with new advances in multi-tilt EM tomography (EMT), chromatin ultrastructure and 3D organization can be visualized at nucleosome resolutions as a continuum through unprecedented nuclear volumes. In Aim 1, ChromEM will be used to visualize chromatin structure-function in human embryonic stem cells and differentiated lung epithelial cells at nucleosome resolutions and genomic scales. We will qualitatively and quantitatively analyze the large-scale changes in chromatin structure and organization in response to Adenovirus infection and HDAC/DNA methyl transferase inhibitors. We will expand the scale and 3D nuclear volumes using serial section EMT and serial block face EM. A major goal will be to reconstruct entire sister chromatid pairs in metaphase cells to determine if they have identical chromosome architectures and chromatin organization. In Aim 2, the EM equivalent of `multi-color' fluorescence will be developed. We will implement `multi-color EM' by using sequential excitation cycles of miniSOG and ChromEM to photo-oxidize different metal chelates of DAB that can be discriminated by elemental mapping using electron energy loss spectroscopy (EELS). Multi-color EM will be used to visualize the structural interactions of viral oncoprotein and PML bodies with chromatin that regulate he silencing and activation of p53 and anti-viral genes. Also, workflow will be extended to incorporate live imaging to visualize the spatiotemporal dynamics of PML and chromatin associated interactions that are disrupted by wild type and mutant adenovirus infections in primary cells. In Aim 3, novel probes will be developed that enable a single copy gene to be identified in a sea of chromatin within the nucleus while preserving native ultrastructure and sequence context. Synthetic self- assembling nanoparticles will be engineered with different architectures, sizes, metal and fluorescent properties so that multiple genes can be labeled and visualized using live imaging, X-Ray microscopy and high resolution 3D EM. These labels will enable GFP-tagged loci to be visualized by EMT together with endogenous gene loci using dCAS9 fusions. The chromatin ultrastructure of telomeres and critical growth regulatory genes will be visualized using live imaging and 3D EM in the dynamics of the cell cycle and viral infection. These studies will change the understanding of the nucleus and chromatin structure-function.